The unusually slow relaxation kinetics of the folding-unfolding of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus

In order to understand the thermodynamic and kinetic basis of the intrinsic stability of proteins from hyperthermophiles, the folding-unfolding reactions of cysteine-free pyrrolidone carboxyl peptidase (Cys142/188Ser) (PCP-0SH) from Pyrococcus furiosus were examined using circular dichroism (CD) and differential scanning calorimetry (DSC) at pH 2.3, where PCP-0SH exists in monomeric form. DSC showed a strong dependence of the shape and position of the unfolding profiles on the scan rate, suggesting the stability of PCP-0SH under kinetic control. On DSC timescales, even at a scan rate of 1 deg. C/hour, heat denaturation of PCP-0SH was non-equilibrium. However, over a long period of incubation of the heat-denatured PCP-0SH at pre-transition temperatures, it refolded completely, indicating reversibility with very slow relaxation kinetics. The rates of refolding of the heat-denatured PCP-0SH determined from the time-resolved DSC and CD spectroscopic progress curves were found to be similar within experimental error, confirming the mechanism of refolding to be a two-state process. The equilibrium established with a relaxation time of 5080 seconds (at t(m)=46.5 degrees C), which is unusually higher than the relaxation times observed for mesophilic and hyperthermophilic proteins. The long relaxation time may lead to the apparent irreversibility of an unfolding process occurring on the DSC experiment timescale. The refolding rate (9.8 x 10(-5) s(-1)) peaked near the t(m) (=46.5 degrees C), whereas the stability profile reached maxima (11.8 kJ mol(-1)) at 17 degrees C. The results clearly indicate the unusual mode of protein destabilization via a drastic decrease in the rate of folding at low pH and still maintaining a high activation energy barrier (284 kJ mol(-1)) for unfolding, which provides an effective kinetic advantage to unusually stable proteins from hyperthermophiles.     

A Simplified Mass-Transfer Model for Visual Pigments in Amphibian Retinal-Cone Outer Segments

When radiolabeled precursors and autoradiography are used to investigate turnover of protein components in photoreceptive cone outer segments (COSs), the labeled components—primarily visual pigment molecules (opsins)—are diffusely distributed along the COS. To further assess this COS labeling pattern, we derive a simplified mass-transfer model for quantifying the contributions of advective and diffusive mechanisms to the distribution of opsins within COSs of the frog retina. Two opsin-containing regions of the COS are evaluated: the core axial array of disks and the plasmalemma. Numerical solutions of the mass-transfer model indicate three distinct stages of system evolution. In the first stage, plasmalemma diffusion is dominant. In the second stage, the plasmalemma density reaches a metastable state and transfer between the plasmalemma and disk region occurs, which is followed by an increase in density that is qualitatively similar for both regions. The final stage consists of both regions slowly evolving to the steady-state solution. Our results indicate that autoradiographic and cognate approaches for tracking labeled opsins in the COS cannot be effective methodologies for assessing new disk formation at the base of the COS.     

SPATIOTEMPORAL VARIATION IN LINEAR NATURAL SELECTION ON BODY COLOR IN WILD GUPPIES (POECILIA RETICULATA)

We conducted 10 mark–recapture experiments in natural populations of Trinidadian guppies to test hypotheses concerning the role of viability selection in geographic patterns of male color variation. Previous work has reported that male guppies are more colorful in low‐predation sites than in high‐predation sites. This pattern of phenotypic variation has been theorized to reflect differences in the balance between natural (viability) selection that disfavors bright male color (owing to predation) and sexual selection that favors bright color (owing to female choice). Our results support the prediction that male color is disfavored by viability selection in both predation regimes. However, it does not support the prediction that viability selection against male color is weaker in low‐predation experiments. Instead, some of the most intense bouts of selection against color occurred in low‐predation experiments. Our results illustrate considerable spatiotemporal variation in selection among experiments, but such variation was not generally correlated with local patterns of color diversity. More complex selective interactions, possibly including the indirect effects of predators on variation in mating behavior, as well as other environmental factors, might be required to more fully explain patterns of secondary sexual trait variation in this system.     

Topological analysis of the complex formed between neurokinin A and the NK2 tachykinin receptor

Neurokinin A stimulates physiological responses in the peripheral and central nervous systems upon interacting primarily with the tachykinin NK2 receptor (NK2R). In this study, the structure of NKA bound to the NK2R is characterised by use of fluorescence resonance energy transfer. Four fluorescent NKA analogues with Texas red introduced at amino acid positions 1, 4, 7 and 10 were prepared. When bound to a NK2R carrying enhanced green fluorescent protein at the N-terminus, all peptides reduce green fluorescent protein fluorescence from 10% to 50% due to energy transfer. The derived donor-acceptor distances are 46, 55, 59 and 69 A for the fluorophore linked to positions 1-10, respectively. The monotonic increase in distance clearly indicates that the peptide adopts an extended structure when bound to its receptor. The present data are used, in combination with rhodopsin structure, fluorescence studies, photoaffinity labelling and site-directed mutagenesis data to design a computer model of the NKA-NK2R complex. We propose that the N-terminus of NKA is exposed and accessible to the extracellular medium. Subsequent amino acids of the NKA peptide become progressively more buried residues up to approximately one-third of the transmembrane-spanning domain.     

The two NK-1 binding sites are distinguished by one radiolabelled substance P analogue

Two non-stoichiometric binding sites had previously been characterized for the NK-1 receptor using two different types of radiolabelled analogues of substance P. However, the question remained on their eventual conformational interconversion induced or not by the ligand. In this study, kinetic, saturation, and competition studies using [3H]propionyl[Pro(9)]SP demonstrate the existence of two independent binding components in CHO cells transfected with the human NK-1 receptor, with K(d) values of 0.040 nM ( approximately 20% of total sites) and 5.9 nM ( approximately 80% of total sites) that correspond to those of the two previously described binding sites. These two binding sites do not seem to interconvert since the minor one can be selectively extinguished in saturation studies in the presence of a SP analogue specific of this binding site.     

Topography and Nanomechanics of Live Neuronal Growth Cones Analyzed by Atomic Force Microscopy

Neuronal growth cones are motile structures located at the end of axons that translate extracellular guidance information into directional movements. Despite the important role of growth cones in neuronal development and regeneration, relatively little is known about the topography and mechanical properties of distinct subcellular growth cone regions under live conditions. In this study, we used the AFM to study the P domain, T zone, and C domain of live Aplysia growth cones. The average height of these regions was calculated from contact mode AFM images to be 183 ± 33, 690 ± 274, and 1322 ± 164 nm, respectively. These findings are consistent with data derived from dynamic mode images of live and contact mode images of fixed growth cones. Nano-indentation measurements indicate that the elastic moduli of the C domain and T zone ruffling region ranged between 3-7 and 7-23 kPa, respectively. The range of the measured elastic modulus of the P domain was 10-40 kPa. High resolution images of the P domain suggest its relatively high elastic modulus results from a dense meshwork of actin filaments in lamellipodia and from actin bundles in the filopodia. The increased mechanical stiffness of the P and T domains is likely important to support and transduce tension that develops during growth cone steering.     

Quantile regression models with multivariate failure time data

As an alternative to the mean regression model, the quantile regression model has been studied extensively with independent failure time data. However, due to natural or artificial clustering, it is common to encounter multivariate failure time data in biomedical research where the intracluster correlation needs to be accounted for appropriately. For right-censored correlated survival data, we investigate the quantile regression model and adapt an estimating equation approach for parameter estimation under the working independence assumption, as well as a weighted version for enhancing the efficiency. We show that the parameter estimates are consistent and asymptotically follow normal distributions. The variance estimation using asymptotic approximation involves nonparametric functional density estimation. We employ the bootstrap and perturbation resampling methods for the estimation of the variance-covariance matrix. We examine the proposed method for finite sample sizes through simulation studies, and illustrate it with data from a clinical trial on otitis media.     

绍鸭卵巢卵泡发育相关新EST的分离与表达

采用银染mRNA差异显示方法从绍鸭卵巢等级卵泡中分离并筛选到 3个差异表达序列标签 (ExpressedSequenceTags ,ESTs)SXDF0 2 0 1(2 71bp)、SXDF0 2 0 2 (2 0 0bp)和SXDF0 2 0 3(173bp) ,通过测序和BLAST检索 ,发现SXDF0 2 0 1与GenBank中登录的所有物种的所有序列均无同源性 ,是在绍鸭卵巢卵泡发现的新EST ,现已登录GenBank(GenBank登录号 :CB0 72 6 2 9) ,而SXDF0 2 0 2和SXDF0 2 0 3则分别与GenBank公布的鸡的EST和肌胃肌球蛋白重链高度同源。用 5′ RACE将SXDF0 2 0 1延伸至 5 4 4bp ,经过BLAST再次检索 ,仍然未发现中度同源序列采用相对定量RT PCR方法进一步研究SXDF0 2 0 1和SXDF0 2 0 2在绍鸭组织中的时空特异性表达 ,发现这两个EST在产蛋高峰期绍兴鸭的下丘脑、垂体、肌肉、肝脏、脂肪等组织都有表达 ;SXDF0 2 0 1在 30日龄卵巢的表达水平显著高于 6 0 (P <0 0 5 )和 90 (P =0 0 15 )日龄 ;而SXDF0 2 0 2在卵巢发育的不同阶段的表达水平没有变化 ;SXDF0 2 0 1在卵巢卵泡颗粒层中的表达总体高于膜层 ,SXDF0 2 0 2在颗粒层中的表达F3 >F5>Fw(P <0 0 1) ,而在F1卵泡降至最低 (P <0 0 1) ,膜层中以Fw 卵泡表达水平最高 (P <0 0 1)。     

Models for age structured populations with distributed maturation rates

In the use of age structured population models for agricultural applications such as the modeling of crop-pest interactions it is often essential that the model take into account the distribution in maturation rates present in some or all of the populations. The traditional method for incorporating distributed maturation rates into crop and pest models has been the so-called distributed delay method. In this paper we review the application of the distributed delay formalism to the McKendrick equation of an age structured population. We discuss the mathematical properties of the system of ordinary differential equations arising out of the distributed delay formalism. We then discuss an alternative method involving modification of the Leslie matrix.